Numerous disorders, including Cystic Fibrosis (CF) and Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency are caused by any one of a number of mutations within the relevant gene.
In 2001, the American College of Medical Genetics (ACMG) and the American College of Obstetrics and Gynecologist (ACOG) recommended that reproductive couples be offered Cystic Fibrosis (CF) screening. CF is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) and is one of the most common autosomal recessive diseases in the North American Caucasian population with an incidence of 1 in 2500-3000 live births (Rommens (1989) and Palomaki (2002)). The carrier frequency of this disease in Northern European, Ashkenazi Jews, Hispanic, African American and Asian descendents is 1 in 25, 1 in 29, 1 in 46, 1 in 65, and 1 in 90, respectively (Grody (2001), Watson (2004)). There are more than 1500 mutations in the CFTR gene as reported by the Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/cftr). Each mutation has various frequencies in different populations. For instance, the mutation DF508 is a mutation that results in deletion of the amino acid phenylalanine at residue 508 and accounts for more than 66% of all CF mutations (Bobadilla, 2002). Cystic Fibrosis has been historically detected by a positive immunoreactive trypsinogen test and more recently by sequencing of the CFTR gene.
ACOG has identified the 23 most common CF causing mutations, which are commonly utilized as the industry standard for genetic testing of CF (see FIG. 1).
Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a condition that prevents the body from converting certain fats to energy, particularly during periods without food (fasting). MCAD deficiency is an inborn metabolic disorder with an incidence of 1 in 10,000 births and can result in death or serious disability. In the United States, the estimated incidence of MCAD deficiency is 1 in 17,000 people. The condition is more common among individuals of northern European ancestry.
People with MCAD deficiency are at risk for serious complications such as seizures, breathing difficulties, liver problems, brain damage, coma, and sudden death. These outcomes can be prevented via diet modification by early diagnosis.
MCAD deficiency is caused by a mutation in the acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain gene (ACADM). More than 80 different mutations in the ACADM gene have been found to cause (MCAD) deficiency. Many of these mutations change single amino acids in the MCAD enzyme. The most common change replaces the amino acid lysine with the amino acid glutamic acid at position 304 in the enzyme (Lys304Glu or K304E). This mutation and other amino acid substitutions alter the enzyme's structure, severely reducing or eliminating its activity. Other types of mutations lead to an abnormally small and unstable enzyme that cannot function.
The presence of a mutation c.985A>G in ACADM exon 11 has been linked to affected phenotype in clinical cases (Matern and Rinaldo, Medium-chain acyl-coenzyme A dehydrogenase deficiency, In: GeneReviews: Genetic Disease Online Reviews at GeneTests-GeneClinics (database online: Initial posting: Apr. 20, 2000; last update Jan. 27, 2003), 2003.). Additional studies have shown the prevalence of this mutation in populations other than Caucasian is low (Matern and Rinaldo). A large newborn screening study performed by the state of New York showed that affected individuals were less likely to be homozygous for the c.985A>G mutation than they were to have other types of mutations such as large deletions or nonsense mutations (Arnold et al. 2010. Lack of genotype-phenotype correlations and outcome in MCAD deficiency diagnosed by newborn screening in New York State. Molecular Genetics and Metabolism 99:263-268). Another large phenotype-genotype study revealed many genetic variants of unknown significance as well as a mutation, c.199T>C in exon 3, that was present in individuals who expressed a milder form of the disease (Smith et al. 2010. Allelic diversity in MCAD deficiency: The biochemical classification of 54 variants identified during 5 years of ACADM sequencing. Molecular Genetics and Metabolism. 100(3):241-50. Epub 2010 Apr 8). These findings suggest that genotype confirmation of MCAD deficiency cannot be limited to detection of a single mutation, and a DNA scanning technique would be useful to rapidly canvass the content of the ACADM gene.
Diagnosis of disorders having a genetic linkage, including CF or MCAD deficiency, or the identification of a carrier individual, requires the analysis of the relevant gene to determine whether a known disease-causing mutation is present. Such analysis may also need to consider whether common variants from the wildtype sequence are present that are not disease-causing.
Methods of DNA analysis including amplification via polymerase chain reaction (PCR), including both standard and asymmetric PCR, and high resolution melting analysis (HRMA) are well known in the art. Recent advances have made such analysis methods available on a microfluidic scale. Description of such advances can be found in, for instance, US 2007/0026241, which is incorporated herein in its entirety.
It has previously been shown that the techniques of scanning (using amplification and HRMA to determine whether a mutation is present, without confirmation of the genotype of the mutation) can be utilized in conjunction with genotyping assays to determine whether mutations are present at a subset of known possible mutation sites and if so, what those mutations are (Zhou et al., “High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution”, Clinical Chemistry 51(10):1770-1777 (2005)). However, this methodology requires that the reactants contain primers for all potential genotypes being tested, and the feasibility of such a method decreases as the complexity of the gene being tested increases. Genes such as CFTR, which is known to have over 1200 mutations or variants, many of which are not recognized as disease causing, would be particularly unsuited to such a method as there would be a high probability of the scanning portion of the assay determining that a mutation or variant was present, without the ability to genotype the mutation unless it was one of the few specifically being tested for. Particularly in regards to common variants that are not disease causing, this method would be inefficient.
There is a need in the art for methods and systems to allow fast, efficient, accurate and cost-effective genetic analysis of DNA samples in order to determine the presence or absence of mutations in a gene of interest. The present application addresses this need.